Prp19 Facilitated p21-Dependent Senescence of Hepatocellular Carcinoma Cells

Introduction Evidence suggests that the role of senescence in the development of cancer is context-dependent. An orthologue of human pre-mRNA processing factor 19 (Prp19) attenuates the senescence of human endothelial cells. Prp19 has been reported to be involved in the progression of hepatocellular carcinoma (HCC). This work aims to investigate the effect of Prp19 on the senescence of HCC. Materials and Methods Senescence of L02 cells and HCC cells under different stimuli was detected through cell cycle analysis, SA-β-gal staining, and senescence associated secretory phenotype analysis. The relationship between Prp19 and senescence-related proteins was evaluated using real-time RT-PCR, western blot assay, and immunohistochemistry. Subcutaneous xenograft tumors in nude mice were used to evaluate the role of Prp19 on senescence in vivo. Data analysis was carried out using GraphPad Prism 6. Results Prp19 facilitated the senescence of L02 cells and HCC cells under different stresses. Prp19 positively modulated p21 expression in the mRNA level. Downregulation of Prp19 promoted the growth of subcutaneous xenograft tumors generated by HCC cell lines. Conclusions Prp19 may promote senescence of HCC cells via regulating p21 expression.


Introduction
Primary liver cancer is the sixth most common cancer in the world and the fourth most common cause of cancer mortality [1]. Globally, hepatocellular carcinoma (HCC) is the dominant type of liver cancer, accounting for approximately 75% of the total [2]. Data from Japan demonstrated that for patients undergoing resection, tumor recurrence, including true recurrence due to dissemination and de novo tumors within the oncogenic liver, complicated 70% of cases at 5 years [3]. Although molecular therapy has shed light on the treatment of patients with advanced HCC, these drugs did not produce significant results [4]. erefore, new targets are pursued in the treatment of HCC.
Cellular senescence, a state of permanent cell cycle arrest, can be induced by multiple stimuli, such as telomere shortening, chromosomal disturbance, oxidative damage, and activation of oncogenes [5]. A large body of evidence has demonstrated a potent tumor suppressor role of senescence in HCC initiation [6][7][8][9]. e mechanisms of senescence in HCC are still elusive. Although senescent hepatocyte-secreted chemokines suppress the initiation of liver cancer, they can accelerate the growth of fully established HCC [10]. erefore the role of senescence in development of HCC is context-dependent. Prosenescence therapy in conjunction with drugs that selectively eliminate senescent HCC cells could be an effective treatment of HCC [11].
Senescence evasion factor, the human orthologue of Prp19, extends the life span of human endothelial cells by increasing resistance to stress [12]. Prp19 is a multifunctional protein involved in many cellular activities [13]. Previous studies have found that Prp19 participates in cellcycle progression and chemotherapeutic resistance of HCC cells [14,15]. Prp19 also facilitated invasion of HCC by promoting twist1-induced epithelial-mesenchymal transition [16]. Considering the important effect of senescence on the development of HCC, the role of Prp19 in senescence during the development of HCC is worth exploring in depth.
In this study, we found a potential prosenescence role of Prp19 on HCC cells. β-Galactosidase (β-gal) staining and senescence associated secretory phenotype (SASP) analysis revealed that Prp19 facilitated senescence in L02 cells and HCC cells under different stresses. Prp19 positively modulated the expression of the senescence-related protein p21 at the transcriptional level. Inhibition of Prp19 promoted tumor growth of HCC cell lines in vivo. CA, USA). Full-length human expression plasmids of human pcDNA3. 1-Prp19 and vector controls were described in our previous work [16]. e H-ras expression plasmid was purchased from Addgene (MA, USA). Plasmid transfection was performed with XtremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany). Cisplatin was obtained from Sigma (Sigma-Aldrich, St. Louis, USA).

Cell Cycle Analysis.
For flow cytometric analysis, cells were collected in phosphate buffered saline (PBS) and fixed in cold ethanol (70-80%) for at least 18 h. Fixed cells were washed three times in PBS and then were stained with propidium iodide (Becton Dickinson, San Jose, CA, USA) for 20 min at 4°C. Finally, cells were analyzed for cell cycle distribution using a BD FACSCalibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). e experiments were independently repeated three times.

SA-β-Galactosidase
Staining. 1 × 10 5 cells were seeded into 12-well plate. Cells under corresponding treatments were rinsed with PBS, fixed with 4% formaldehyde in PBS, and then incubated with freshly prepared senescence-associated β-gal staining solution (Beyotime, Nantong, China) at 37°C overnight. β-gal positive cells were counted in randomly selected fields per 100 cells. Frozen xenograft tumors were evaluated for in vivo assay.

Real-Time RT-PCR, Western Blotting Assay, and
Immunohistochemistry.
e total RNA of HCC cells was extracted by TRIzol (Invitrogen, NY, USA). e synthesis of the first strand of cDNA was performed with the AMV RNA PCR kit (TaKaRa, Dalian, China) following the manufacturer's protocol. Subsequent real-time PCR was performed using a SYBR Green Premix Ex Taq (TaKaRa, Dalian, China) on the ABI StepOne Plus system (Applied Biosystems, CA, USA). e primers used in real-time RT-PCR are listed in Supplementary Table 2. Western blot assay was performed to analyze cell lysate extracts using ImageQuant LAS 4000mini (GEHealthcare, New Jersey, USA). e antibodies used are listed in Supplementary Table 3. Immunohistochemistry of paraffin-embedded sections was performed as reported elsewhere [17].
2.6. Animal Experiments. BALB/C nude mice (4 weeks old, male) were obtained from the Shanghai Institute of the Material Medicine of Chinese Academy of Science. Stable HCC cells misexpressing Prp19 were injected subcutaneously into the left armpit of nude mice (n � 5). e size of subcutaneous xenograft tumors was measured weekly after injection. Four weeks later, xenograft tumors were prepared for hematoxylin and eosin staining, immunohistochemistry analysis, and β-gal staining. Animal care and experimental protocols were approved by the Experimental Animal Care Commission of Shanghai Medical School of Fudan University (Shanghai, China) and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Statistical
Analysis. GraphPad Prism 6 (La Jolla, CA, USA) was used for data analysis. Data were presented as the mean ± SEM of three independent studies. Student's t-test was used to compare differences between different groups. P value less than 0.05 was recognized as statistically significant. * p < 0.05, * * p < 0.01, and * * * p < 0.001.

Prp19 Facilitates Senescence of L02 Cells.
Our previous study suggested that Prp19 arrested the cell cycle of HCC cells at the G1 phase [14], we speculated that Prp19 may have an effect on senescence. Since the protein level of Prp19 in normal hepatocyte L02 was lower than that in HCC cells [16], Prp19 was overexpressed in L02 cells to evaluate the impact of Prp19 on the senescence of normal hepatocyte (Figure 1(a)). Upregulating Prp19 expression in L02 cells resulted in G1 arrest (Figure 1(a)), while more β-gal positive cells were observed in L02 cells overexpressing Prp19 compared with vector controls (Figure 1(b)). We also found that Prp19 promoted cisplatin-induced senescence of L02 cells (Figure 1(b)). Furthermore, overexpression of the H-ras oncogene in L02 cells led to senescence, while upregulation of Prp19 promoted H-ras-induced senescence in L02 cells (Figure 1(c)). Taken together, these results demonstrated that Prp19 facilitated the senescence of L02 cells with or without stimuli.

Prp19 Modulates p21 Expression in L02 Cells and HCC
Cells. Western blot assay showed that p21 rather than p53 or p16 was increased in L02 cells overexpressing Prp19 (Figure 3(a)). Prp19 knockdown in HCC cells showed a robust reduction in p21 protein expression, while silencing Prp19 did not affect p53 or p16 protein expression, two other important senescence modulators (Figure 3(b)). Considering the vital role of Prp19 in pre-mRNA processing, we assessed p21 mRNA levels in HCC cells with Prp19 downregulation. Real-time RT-PCR analysis showed that silencing Prp19 significantly decreased p21 mRNA levels in HCC cells compared with controls ( Figure 3(c)). ese findings suggested that Prp19 may participate in the senescence of HCC cells by regulating the expression of p21 mRNA.

Prp19 Knockdown Promotes In Vivo Tumor Growth of HCC Cells.
Senescence is an important barrier against cell proliferation. A previous study showed that Prp19 had no significant impact on HCC cell proliferation in vitro [16]. To investigate the role of Prp19 on tumor growth in vivo, we generated two stable HCC cell lines misexpressing Prp19 and then established subcutaneous xenograft models of HCC. Compared to the NTC groups, Prp19 expression was attenuated in the shPrp19 groups (Figure 4(a)). In this model, tumor volume was measured weekly. e mean tumor volume and weight in the NTC groups were significantly decreased compared to the shPrp19 groups (Figures 4(b) and 4(c)). In the shPrp19 groups, the expression of Prp19 and p21 was decreased in xenograft tumor tissues compared to the NTC groups (Figures 4(d) and 4(e)). Unlike the NTC group, there were fewer senescent areas in the xenograft tissues of the shPrp19 group (Figures 4(d) and 4(f )). Abovementioned data suggested that Prp19 knockdown inhibited HCC cell senescence and promoted tumor growth of HCC cells in vivo.

Discussion
Senescence is an important cellular response to various stimuli such as oncogene activation as well as exposure to DNA damage. Prp19 was reported to reduce DNA damageinduced senescence in mouse skin and increase the resistance to stress in human endothelial cells [12,18]. Mouse embryonic fibroblast cell lines with reduced Prp19 levels displayed a decreased proliferative potential in vitro [19].
e abovementioned studies implied that Prp19 may modulate senescence. Aging is a major risk factor for the progression of liver disease to HCC [20]. Cirrhosis is characterized by replicative senescence due to short telomeres and no expression of telomerase in mature hepatocytes [21]. is study found that Prp19 promoted L02 cells senescence under different stimuli, including DNA damage exposure and oncogene activation. Taking into account the gradual elevation of Prp19 expression from normal liver to HCC in a previous study [16], this finding indicated the underlying role of Prp19 in senescence during the development of liver cirrhosis.
Prp19 expression is abundant in many human cancers, including HCC [16,22,23]. It was reported that Prp19 repressed cell proliferation and tumor growth via upregulating the protein expression of p21 [23]. We also identified the role of Prp19 in the progression of the cell cycle in HCC [14]. Recently, mechanisms behind senescence and druginduced senescence have been partially unveiled in HCC [9,20,24]. is work indicated that Prp19 downregulation inhibited senescence as well as cisplatin-induced senescence in HCC cells. Although no significant impact of Prp19 on the proliferation of HCC cells was observed in vitro, we found that repression of Prp19 in HCC cells facilitated tumor growth in vivo. It was demonstrated that the secretion of the interleukins IL-6, IL-8, and CXCL-1 increased oncogeneinduced growth arrest caused by Ras and Braf [25,26]. In this study, Prp19 down-regulation resulted in the decreased mRNA expression of SASP factors such as CXCL-1, IL-6, and IL-8 in HCC cells, which may account for the proliferation of tumor with Prp19 deficiency. A recent study reported that while senescent hepatocyte-secreted chemokines suppressed liver cancer initiation, they may accelerate the growth of fully established HCC [10]. Since the pro or antitumorigenic effect of senescence depends on the context, the promoting role of Prp19 on the progression of HCC may be partly owing to its impact on senescence.
It is well recognized that the p53-p21 and the p16-RB pathways are critical for senescence [27]. Among these important regulators of senescence, we found Prp19 positively modulated p21 expression rather than p53 or p16 expression in L02 cells and HCC cells. Prp19 silencing was reported to comprise p21 mRNA splicing [28]. Here, we found that inhibiting Prp19 expression decreased the p21 mRNA level in HCC cells. is study did not observe the modulation of Prp19 in p53 expression, and the molecular 4 Journal of Oncology  Journal of Oncology mechanism underlying Prp19-induced alteration of p21 needs further study in the future. In summary, this work first revealed the positive role of Prp19 on senescence in L02 cells and HCC cells under different stimuli. Furthermore, we found that Prp19 robustly modulated p21 expression in HCC cells. Repression of Prp19 resulted in a reduction of senescence and acceleration of tumor growth in vivo. It was reported that inhibition of Prp19 made HCC cells less resistant to chemotherapeutic agents [15]. Compromised senescence in HCC cells with Prp19 deficiency may drive cell cycle progression, resulting in increased chemotherapeutic sensitivity, rendering Prp19 a potential target in HCC management.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
is study is in compliance with ethical standards.

Disclosure
In 2020, an earlier version of this manuscript's abstract was presented as a conference abstract in the WILEY Online Library.